The present invention relates to magnetic resonance imaging, and more particularly to a method for preparing magnetic resonance imaging agents.
Magnetic resonance imaging (MRI) is an important technique for detecting abnormalities in a patient's body, thereby aiding in the diagnosis and treatment of certain diseases. MRI is advantageous because it is noninvasive, and there is no exposure to harmful radiation. Using MRI, normal and diseased tissues may be differentiated on the basis of differences in relaxation times, T1 or T2 wherein T1 refers to the spin-lattice or longitudinal relaxation time, and T2 refers to the spin-spin or transverse relaxation time. In some cases, however, the abnormal tissues have the same relaxation times as the normal tissues.
MRI contrast-enhancing agents enhance various portions of the MR image by changing, usually increasing, the relaxation rate of bulk water protons in close proximity to the agent. Thus, the imaging contrasts between normal and abnormal tissues are enhanced. Low molecular weight contrast-enhancing agents, such as gadolinium (III) diethylenetriaminepentaacetic acid (Gd.DTPA) (MW=538), have been widely used for this purpose because they can diffuse rapidly into plaques. However, due to their small molecular size, they also tend to clear away rapidly from the body. Thus, the imaging procedure must be completed within a very short time after such agents are administered into a patient. In addition, these low molecular-weight agents deliver only a limited number of contrast-enhancing ions to the region of interest.
The introduction of high molecular weight MRI contrast-enhancing agents using natural and synthetic macromolecules into the circulation remain longer, providing ample time to migrate across the tumor endothelium. These agents also easily diffuse through the endothelial layer of the tumor. Such agents comprise multiple chelating groups, such as DTPA, coupled to a singular polymer backbone, such as a poly(amino acid). These chelating moieties also form coordination complexes with paramagnetic cations, such as gadolinium (III), dysprosium (III), holmium (III), europium (III), iron (III), or manganese (II). For example, poly(1-lysine-Gd.DTPA), has been shown to be an excellent MRI contrast-enhancing agent.
These high molecular weight contrast-enhancing agents bearing a poly(amino acid) backbone conjugated to chelating moieties complexed with paramagnetic ions demonstrate selective association and imaging of diseased tissue making them useful for detecting tumors, for example. The tissue selectivity and imaging intensity of these agents is believed to rely on a high degree of conjugation of the poly(amino acid) repeat units with the gadolinium (Gd) (or other paramagnetic ion) chelator. However, highly conjugated polymers, such as conjugated poly(1-lysine-Gd.DTPA), are difficult to synthesize reliably.
Briefly, as described by Sieving et al. in Bioconjugate Chem. 1, 65–71 (1990), the standard procedure for the synthesis of poly(1-lysine-Gd.DTPA) involves coupling of DTPA to the preformed poly(1-lysine) polypeptide followed by incorporation of the gadolinium ion (in the form of a salt, such as GdCl3) into the poly(1-lysine-DTPA) intermediate. However, this method of preparing poly(1-lysine-DTPA) suffers from variable conjugation efficiency, which is preferably >90% lysine repeat units functionalized with DTPA, and requires an extended purification procedure to remove by-products.
Furthermore, the current synthetic methodology for poly(1-lysine.DTPA) calls for the monoactivation of DTPA by conversion to a carboxy anhydride reactive intermediate, where multiactivation of DTPA can lead to poly(1-lysine) crosslinking and consequently to a loss in imaging selectivity. Also, as previously stated, the by-products of the DTPA poly(1-lysine) coupling reaction require a lengthy and tedious separation, resulting in a poorer product yield.
Related co-pending commonly owned U.S. patent applications Ser. No. 10/609,269 filed Jun. 26, 2003 and 10/209,726 filed Jul. 31, 2002 (published Feb. 5, 2004 under Publication No. US 2004/0022857) disclose efficient means of preparing extended poly(amino acids) conjugated to chelator moieties that form coordination complexes with paramagnetic ions. The method involves the low temperature activation of DTPA.5NEt3, wherein NEt3 is triethylamine, with isobutylchlorofomate (IBCF) in order to maximize the amount of monoactivated DTPA produced. While this approach provides additional efficiencies based upon its use of lower temperatures relative to known methods, improved methods continue to be sought, especially in light of the challenges presented for synthesizing highly conjugated polymers.